Field of Disclosure
This disclosure relates generally to a method for the biosynthetic production of capsaicin and related capsaicinoids, particularly utilizing acyl-CoA synthetase (ACS), aminotransferase (pAMT) and capsaicin synthase (CS).
Background Art
The chili pepper is the fruit from plants of the genus Capsicum, members of the nightshade family, Solanaceae. Chili pepper has been widely used as a food additive in spicy and hot cuisines, due to its pungent nature. Capsacinoids are the substances responsible for the pungent sensation of the chili pepper and as mentioned previously, their production is restricted to the genus Capsicum. Capsaicin (CP, 8-methyl-N-vanillyl-trans-6-nonenamide) and dihydrocapsaicin (DHCP, 8-methyl-N-vanillylnonanamide) are the two major capsaicinoids responsible for roughly up to 90% of the pungency in chili pepper (Garcés-Claver, et al., 2007).
In addition to being used mainly as food additives for hot sensation and spicy flavoring, capsaicinoids have many pharmaceutical and medical uses. They have been found to exert a series of physiological and pharmacological effects, including analgesia, anti-cancer, anti-inflammatory, anti-oxidative and anti-obesity activities and are used as the main components ointments, patches, oils and creams designed to relieve the pain caused by several diseases such as vasomotor rhinitis, osteoarthritis and rheumatoid arthritis (Aza-González, et al., 2011). Capsaicinoids are also currently used as the main active ingredient in self-protective aerosol sprays (i.e., pepper sprays) on the market (Reilly, et al., 2001). Recently capsaicinoids were reported to lower plasma cholesterol and improve endothelial function in hamsters (Liang, et al., 2013).
Capsaicin is believed to be synthesized by CS, an acyltransferase that transfers the 8-methylnonenoyl moiety from 8-methylnonenoyl-CoA to vanillylamine to form an amide conjugate (FIG. 1). Vanillylamine is formed from the phenylpropanoid pathway wherein the branched-chain fatty acid is derived from a branched-chain amino acid, e.g., valine (Curry, et al., 1999; Mazourek, et al., et al., 2009). The aminotransferase (pAMT) catalyzes the formation of vanillyamine from vanillin. Applicants have cloned pAMT derived from ghost chili pepper. The other substrate, 8-methylnonenoyl-CoA, is derived from 8-methyl-trans-6-nonenoic acid through the activity of an acyl-CoA synthetase (ACS).
In this disclosure, applicants have utilized the gene product of CS to produce capsaicinoids by microbial biosynthesis. Applicants are the first to achieve microbial production of capsaicinoids, particularly capsaicin. Moreover, this invention addresses a long-felt but unmet need in the industry to produce capsaicin by microbial biosynthesis.